P12.50.A UNLOCKING 3D INSIGHTS: ENHANCED SURVIVAL AND IMAGING METHODS FOR HUMAN CORTICAL SLICE CULTURES

Abstract BACKGROUND Human cortical slice cultures represent valuable ex vivo models, yet achieving robust long-term survival and enabling effective 3D tissue analysis remain key challenges. This study aimed to establish improved protocols addressing viability, tissue processing, and comprehensive 3D staining and imaging for these complex tissues. MATERIAL AND METHODS Human cortical slices (300µm) were cultured over 14 days comparing four media: our established growth medium (GM), a new adapted variant (GMN), aCSF, and pooled hCSF. Analysis was performed at five time points (D0, D1, D4, D7, D14). The FFPE embedding protocol was improved to preserve tissue integrity for sectioning. For quantification, FFPE sections (5µm) underwent multiplex immunofluorescence (IF) staining measuring Bcl-2 (apoptosis inhibitor) and cell markers (NeuN, Iba1, GFAP, Olig2, MBP, PDGFRβ, CD31) with regional analysis and IHC verification. Separately, a novel free-floating IF staining protocol was developed for whole-mount staining of intact slices. High-resolution 3D imaging of these whole-mount stained slices was performed using a Leica Stellaris 8 confocal microscope with Imaris reconstruction. Single-nucleus RNA sequencing (snRNA-seq) was performed. These methods were applied to both control and tumor-infiltrated slices. RESULTS Analysis of multiplex IF stainings revealed that pooled hCSF provided the highest long-term viability, followed sequentially by aCSF, GMN, and GM, based on quantitative Bcl-2 analysis. The novel free-floating whole-mount staining protocol achieved uniform antibody penetration throughout intact 300µm slices, allowing characterization of cellular dynamics, including subtle morphological changes in microglia (Iba1+) between D0 and D1. It also facilitates detailed 3D visualization of cellular architecture in combination with retrograde tracing signals from injected tumor cells. This whole-mount approach preserves 3D context and increases analytical efficiency compared to serial sectioning. High-resolution 3D reconstructions were generated from Leica Stellaris 8 datasets. snRNA-seq profiles correlated with observed phenotypes and survival across media conditions. CONCLUSION This work establishes optimized protocols for enhancing survival, improving tissue processing, and enabling comprehensive analysis of human cortical slice cultures. The developed whole-mount staining technique allows for detailed 3D investigation of cellular interactions and architecture in thick brain tissues. These methods provide a powerful platform for advancing neuro-oncological research and modeling complex processes, such as tumor microenvironment interactions, within a clinically relevant human system.